pNTAP-PRAK真核表达载体的构建及其在HEK293细胞中的表达

目的: 构建pNTAP-PRAK真核表达质粒,并建立其稳定表达的HEK293细胞系。方法: 将人的PRAK亚克隆至串联亲和纯化(tandem affinity purification, TAP)载体pNTAP质粒上,构建成重组质粒pNTAP-PRAK,转化该重组质粒至感受态大肠杆菌DH5α,阳性克隆进行PCR、酶切及DNA测序验证正确后,利用PolyFect脂质体介导将其转染至HEK293细胞中,再通过G418筛选建立稳定表达TAP tag-PRAK融合蛋白的HEK293细胞系;利用Western blotting和细胞免疫荧光标记法检测融合蛋白TAP tag-PRAK的表达及细胞内定位情况。结果: 重组真核表达载体构建正确,该重组质粒能在HEK293细胞中稳定表达,表达产物TAP tag-PRAK主要分布在核内。结论: 成功构建pNTAP-PRAK真核表达载体并建立了其稳定表达的HEK293细胞系,TAP标签未对PRAK定位产生明显影响。     

转染腺病毒载体对雌二醇促垂体催乳素细胞增殖的影响

目的探讨转染腺病毒载体对雌二醇促垂体催乳素细胞增殖的影响及可能的分子机制。方法应用BrdU标记法检测转染腺病毒载体后雌二醇促垂体细胞增殖作用的变化,实时荧光定量PCR方法检测腺病毒载体转染组雌激素反应基因ABCG2的mRNAs表达水平,Western Blot方法检测腺病毒载体对垂体前叶细胞磷酸化p38MAPK蛋白表达的影响。结果转染腺病毒载体显著下调了雌二醇的促催乳素细胞增殖作用(P0.01),腺病毒转染组磷酸化p38MAPK蛋白表达显著增加,ABCG2基因的表达显著下降(P0.01)。结论腺病毒载体转染下调雌二醇促垂体催乳素细胞的增殖作用,其机制可能与腺病毒载体改变p38MAPK和ABCG2的表达有关。     

Segregation of non‐p.R132H mutations in IDH1 in distinct molecular subtypes of glioma

Mutations in the gene encoding the isocitrate dehydrogenase 1 gene (IDH1) occur at a high frequency (up to 80%) in many different subtypes of glioma. In this study, we have screened for IDH1 mutations in a cohort of 496 gliomas. IDH1 mutations were most frequently observed in low grade gliomas with c.395G>A (p.R132H) representing >90% of all IDH1 mutations. Interestingly, non‐p.R132H mutations segregate in distinct histological and molecular subtypes of glioma. Histologically, they occur sporadically in classic oligodendrogliomas and at significantly higher frequency in other grade II and III gliomas. Genetically, non‐p.R132H mutations occur in tumors with TP53 mutation, are virtually absent in tumors with loss of heterozygosity on 1p and 19q and accumulate in distinct (gene‐expression profiling based) intrinsic molecular subtypes. The IDH1 mutation type does not affect patient survival. Our results were validated on an independent sample cohort, indicating that the IDH1 mutation spectrum may aid glioma subtype classification. Functional differences between p.R132H and non‐p.R132H mutated IDH1 may explain the segregation in distinct glioma subtypes. © 2010 Wiley‐Liss, Inc.     

先天性缺指（趾）-外胚层发育不良-唇/腭裂综合征的临床研究

目的探讨先天缺指（趾）-外胚层发育不良-唇/腭裂综合征的临床表现,诊断标准,遗传学特点及治疗措施。方法 2008年3月至2009年9月收集先天缺指（趾）-外胚层发育不良-唇/腭裂综合征女性患者一例,针对上唇缺损行手术修补,对其进行家系问卷和DNA检查,观察患者的临床表型和发病特点,分析可能的遗传方式。结果术后患者伤口愈合良好,无并发症发生,此例患者随访半年,术后明显改善了患者的外观,研究收集的这个典型病例未追溯到明显家族遗传史。结论收集的一个患者属典型的散发病例,通过对该患者的诊疗,证明治疗应是多学科的,主要是针对外观进行整形外科手术,临床的早期检查和正确诊断对后期治疗具有重要意义。产前诊断意义尤为重大。     

Down-regulation of p38 mitogen-activated protein kinase activation and proinflammatory cytokine production by mitogen-activated protein kinase inhibitors in inflammatory bowel disease

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)‐α, a known agonist of the mitogen‐activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho‐p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex‐ and age‐matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF‐α, interleukin (IL)‐1β and IL‐6 were measured in the organ and cell culture supernatants by enzyme‐linked immunosorbent assay. We found higher levels of phospho‐p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF‐α, IL‐1β and IL‐6 from IBD LPMCs and biopsies. Activated p38α MAPK is up‐regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down‐regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.     

Primary squamous cell carcinoma of the vagina: human papillomavirus detection, p16(INK4A) overexpression and clinicopathological correlations

Fuste V, del Pino M, Perez A, Garcia A, Torne A, Pahisa J & Ordi J
(2010) Histopathology 57, 907–916 Primary squamous cell carcinoma of the vagina: human papillomavirus detection, p16 ^INK4A overexpression and clinicopathological correlations Aim: To determine the role of human papillomavirus (HPV) in the pathogenesis of primary squamous cell carcinoma of the vagina (SCCVa), and to evaluate its clinicopathological significance. Methods and results: All cases of SCCVa diagnosed over a 15‐year period from two hospitals in Barcelona, Spain (n = 32) were retrieved. Patients with a history of carcinoma of the cervix diagnosed <5 years before were excluded. HPV was detected and typed by polymerase chain reaction (PCR) using SPF10 primers. Immunohistochemistry was performed for p16 and p53. HPV was detected in 25 cases (78.1%). HPV16 was the most prevalent type. Patients with HPV‐positive tumours were associated frequently with a history of carcinoma or intraepithelial neoplasia of the cervix or vulva diagnosed more than 5 years before (56% versus 0%; P = 0.01). HPV‐positive tumours were more frequently of non‐keratinizing, basaloid or warty type than HPV‐negative neoplasms (84% versus 14.3%; P < 0.001), and showed diffuse positive immunoreactivity for p16^INK4a (96%, versus 14.3%; P < 0.001). The sensitivity and specificity of p16 to identify HPV‐positive tumours were 96% and 85.7%, respectively. Conclusions: A high number of SCCVs are related to HPV infection and may be identified by immunohistochemistry for p16. HPV‐positive tumours tend to affect women with history of cervical neoplasia.     

Non-receptor tyrosine kinase Etk is involved in the apoptosis of small cell lung cancer cells

Epithelial and endothelial tyrosine kinase (Etk), also known as Bmx (bone marrow X kinase) plays an important role in apoptosis of epithelial cells. The goal of this study was to investigate whether Etk is involved in apoptosis of small cell lung cancer (SCLC) cells and correlated with the expression levels of apoptosis-associated proteins such as Bcl-2, Bcl-X_L and p53. One hundred and seventy-one cases of lung cancer specimens including seventy-one SCLCs and one hundred NSCLCs were immunostained for Etk, Bcl-2, Bcl-X_L and p53. Parental SCLC H446 cell line, and its subline (H446-Etk) that overexpresses Etk, were used to study the role of Etk in apoptosis induced by doxorubicin. It was found that high expression of Etk occurs in 74.6% of SCLC cases, but only in 40% of NSCLC cases, and there is marked difference in the expression levels of Bcl-2, Bcl-X_L and p53 between Etk-positive and Etk-negative SCLC cases. Furthermore, the levels of Bcl-2 and Bcl-X_L significantly increased in H446-Etk cells than that in H446 cells after doxorubicin treatment, and were positively associated with Etk expression. However, p53 did not correspond with Etk expression although its expression decreased greatly with apoptosis both in H446-Etk and H446 cells. After doxorubicin treatment, the cell viability was significantly higher in H446-Etk cells than in parental H446 cells. Downregulation of Etk by Etk siRNA sensitized H446 cells to doxorubicin. Our results indicate that upregulation of tyrosine kinase Etk may be a new mechanism involved in protection of SCLC cells from apoptosis. Bcl-2 and Bcl-X_L but not p53 may contribute to doxorubicin-induced apoptosis through Etk pathway.     

鼻咽癌中p27kip1表达及其与临床病理特征相关研究

目的 检测p27kip1蛋白在鼻咽癌中的表达,分析其与临床病理特征的关系.方法 收集60例手术活检切取的鼻咽癌组织蜡块,与30例非肿瘤鼻咽组织石蜡标本作比较.应用免疫组织化学技术检测鼻咽癌组织中p27kip1蛋白的表达,回顾性研究鼻咽癌患者的临床病理特征.结果 p27ksp1蛋白在鼻咽癌细胞核和细胞质中均有表达.鼻咽癌组织中细胞核表达阳性率为46.7%(28/60),低于非肿瘤鼻咽组织细胞核表达的90%(27/30),P＜0.01;而在细胞质中阳性率为68.3%(41/60),显著高于非肿瘤鼻咽组织的细胞质表达的20%(6/30),P＜0.01.未发现p27kip1蛋白在细胞核和细胞质表达与鼻咽癌原发灶的范围和局部侵犯程度有关,但p27kip1蛋白胞核表达与淋巴结转移、远处转移和TNM临床分期有关;细胞质表达仅与淋巴结转移和TNM临床分期有关,可见p27kip1蛋白胞核表达与临床病理的关系更为密切.结论 与非肿瘤鼻咽组织相比,p27kip1蛋白为细胞核低表达、细胞质高表达.鼻咽癌中p27kip1蛋白存在不同于非肿瘤鼻咽组织的错误的核质定位,其在细胞核中表达的减少或丢失可能与鼻咽癌的恶性发展有关,提示p27kip1蛋白的异常表达和定位可能涉及鼻咽癌的分期和转移.     

优秀临床医师的人格特征

目的了解优秀医师的人格特点。方法通过卡特尔16种人格因素问卷对160名临床医师进行测查,使用SPSS软件分析优秀医师和对照组的人格特点差异。结果优秀男医师的A(t=2.912,P0.05)、C(t=2.103,P0.05)、F(t=7.031,P0.01)、Q2(t=-2.221,P0.05)、Q3(t=2.501,P0.05)5个因子与男临床医师存在显著性差异;优秀女医师的A(t=6.308,P0.01)、C(t=3.077,P0.01)、F(t=7.258,P0.01)、G(t=2.315,P0.05)、H(t=4.269,P0.01)、O(t=-2.596,P0.05)、Q3(t=2.227,P0.05)7个因子与女临床医师存在显著性差异。结论男女优秀临床医师在16PF的A(乐群性)、C(稳定性)、F(兴奋性)、Q3(自律性)4个因子上与对照组相比均表现出高分特征,这些特质可能与临床医师的工作绩效相关。     

The human papillomavirus type 16 E5 oncoprotein synergizes with EGF-receptor signaling to enhance cell cycle progression and the down-regulation of p27

E5 oncoprotein activity from high risk human papillomaviruses (HPVs) is associated with growth factor receptor signaling, but the function of this protein is not well understood. In this study, we investigated the role of HPV-16 E5 on the cell cycle progression during EGF-stimulation. Wild-type and NIH 3T3 cells over-expressing human EGF-receptor were transfected with HPV-16 E5 gene and the cell cycle progression was characterized. This analysis showed that the E5-expressing cells increased DNA synthesis (S-phase) by around 40%. Cell cycle protein analysis of E5-expressing cells showed a reduction in the half-life of p27^Kip1 protein as compared to control cells (18.4 vs. 12.7 h), an effect that was enhanced in EGF-stimulated cells (12.8 vs. 3.6 h). Blockage of EGF-receptor activity abrogated E5 signals as well as p27^Kip1 down-regulation. These results suggest that E5 and the EGF-receptor cooperate to enhance cell cycle entry and progression through regulating p27^Kip1 expression at protein level.